In the preparation of human insulin by genetic engineering methods, biosynthesis of the insulin is carried out via a precursor molecule called proinsulin. In this, the B chain is linked to the A chain by the C peptide (=connecting peptide). In genetic engineering production by means of Escherichia coli, a fusion protein in which another foreign protein, for example .beta.-galactosidase, precedes the proinsulin is sometimes first obtained. This foreign protein must first be split off, for example by treatment with a cyanogen halide, before further working up (compare German Offenlegungsschrift 34 40 988). After the cyanogen halide cleavage, cysteine radicals are converted into their S-sulfonate form by sulfitolysis (treatment with sodium sulfite and sodium tetrathionate). The molecule can be converted from this form into its natural spatial structure with correct formation of its disulfide bridges by reductive folding back (for example by treatment with mercaptoethanol in basic solution). The proinsulin or preproinsulin (=derivative of proinsulin; the prefix "pre" relates to one or more additional amino acids on the N terminus of the proinsulin) is converted by enzymatic cleavage (for example with trypsin) into a cleavage mixture which contains an insulin precursor, insulin-Arg-B31-B32, and the C peptide. In addition to these, some by-products, such as, for example, insulin-Des-Thr-B30, insulin-Arg-B31 and incompletely cleaved intermediates, are also formed.
The insulin derivatives mentioned and compounds which are formed in preliminary stages by chemical treatment of the starting material must be separated from one another.
It is particularly difficult here to separate insulin derivatives of basic character which have derivatizations on amino acids which, after formation of the tertiary structure, lie inside the molecule.
This is found in a comparison experiment (see Part B): the anion exchanger process which has been described in German Patent 26 29 568 and has been developed for purification of insulin has the peculiarity that a nonionic surfactant is added in order to avoid protein aggregations of the elution liquid. This process, which is particularly suitable for the purification of insulins, leads to a completely inadequate separation in the attempt to separate and isolate basic proteins which are obtained, for example, by tryptic cleavage of proinsulin of genetic engineering origin. This is illustrated by the corresponding elution profile (see Part B); it can be seen that the peaks for the individual peptides overlap one another.
Processes are also already known which are said to achieve separation of the proinsulin cleavage products mentioned. Steiner et al. describe in "Journ. of Biol. Chem.", 246, pages 1365-1374 (1971) a process for isolating C peptide from a proinsulin cleavage mixture. In this process (no yields are stated), chromatography is carried out over a weakly acid cellulose-based cation exchanger containing carboxymethyl groups (CM). 7 mol/l of urea are added here to the loading and elution solution in order to avoid aggregation of the proteins. The presence of such high urea concentrations is a disadvantage because it leads to derivatization of proteins, above all to carbamoylation of free amino groups. Markussen et al. describe in "Protein Engineering" Volume 1 No. 3, pages 205-213 (1987) an anion exchanger chromatography process on diethyl-2-hydroxypropylaminoethyl-containing (QAE) anion exchangers on a matrix of a three-dimensional crosslinked polysaccharide network.
This process--no yields are given for the anion exchange chromatography--is carried out in 60% strength ethanol solution to inhibit protein aggregations. The safety measures required, which are particularly necessary when working with concentrated organic solvents on an industrial scale, are a disadvantage of ethanol solutions of such a concentration. On the other hand, as reworking has shown, denaturations of the proteins occur at the alcohol concentrations mentioned.